We have shown previously that RhBG & RhCG are novel homologues of the red cell Rh antigens. This project centers on the functional role and mode of action of RhBG & RhCG, with the long-term goals to elucidate their substrate specificity, differential expression and physiological regulation in mammalian nonerythroid tissues (kidney, liver, skin, brain, testis and ovary). The proposed studies are based on our findings concerning their primary sequence features, gene structure, tissue specificity, epithelial membrane location, protein interaction and conserved evolution from unicellular organisms to man. With these data, we can now address the function of Rh family proteins, clarifying whether they are ammonia transporters (Amt), CO2 channels or plasma membrane-remodeling proteins. We postulate: 1) RhBG & RhCG are structurally connected to microbial Amts but functionally orthologous to the Rh proteins of primitive organisms. 2) RhBG & RhCG may not transport ammonium but act similarly in a channel-type mechanism mediating CO2 diffusion and facilitating CO2 conversion into certain organic forms via functionally associated proteins. 3) RhBG & RhCG require specific protein interactions to function properly, and their differential expression is regulated at the level of gene transcription and splicing. 4) In complex with their partners, RhBG & RhCG execute related but distinct physiologic roles in specific renal sites arid target organs. These hypotheses will be tested using mammalian kidney cells and tissues as a major model. The four specific aims are as follows: 1) resolve the distribution of RhBG & RhCG in the various renal segments, and establish whether they exhibit polarized location in the apical or basal membrane; 2) define the molecular basis for the differential expression and regulation of RhBG vs. RhCG gene by dissecting their promoter activity and splicing pattern; 3) identify the cellular components physically associated with RhBG or RhCG in renal tissues, and characterize their interactions by cell, biochemical or genetic approaches; 4) determine the substrate specificity of RhBG/RhCG and associated proteins, and elucidate the mode of their action as to whether the Rh proteins and the partner(s) function in parallel or cooperatively. The results obtained should contribute to understanding the structure, function and regulation of the Rh family, revealing a potentially important, new homeostatic mechanism operative in human and mammalian organs.